Targeting HSV-1 virions for specific binding to epidermal growth factor receptor-vIII-bearing tumor cells

Abstract

Oncolytic herpes simplex virus (HSV) vectors have been used in early phase human clinical trials as a therapy for recurrent malignant glioblastoma. This treatment proved safe but limited improvements in patient survival were observed. The potency of these vectors might be enhanced by targeting vector infectivity to tumor cells. Glioma tumors often express a mutant form (vIII) of the epidermal growth factor receptor (EGFR) resulting in the presence of a novel epitope on the cell surface. This epitope is specifically recognized by a single-chain antibody designated MR1-1. HSV-1 infection involves initial binding to heparan sulfate (HS) on the cell surface mediated primarily by the viral envelope, glycoprotein C (gC). Here we joined the MR1-1 single-chain antibody (scFv) to the gC sequence deleted for the HS-binding domain as a means of targeting viral attachment to EGFRvIII on glial tumor cells. Virions bearing MR1-1-modified gC had fivefold increased infectivity for EGFRvIII-bearing human glioma U87 cells compared to mutant receptor-deficient cells. Further, MR1-1/EGFRvIII-mediated infection was more efficient for EGFRvIII-positive cells than was wild-type virus for either positive or negative cells. Sustained infection of EGFRvIII+ glioma cells by MR1-1-modified gC-bearing oncolytic virus, as compared to wild-type gC oncolytic virus, was also shown in subcutaneous tumors in vivo using firefly luciferase as a reporter of infection. These data show that HSV tropism can be manipulated so that virions recognize a cell-specific binding site with increased infectivity for the target cell. The retargeting of HSV infection to tumor cells should enhance vector specificity, tumor cell killing and vector safety.

Fig. 1. MR1-1-gCΔ construct

On the let panel, structure of HSV-amplicon vector pCONG-MR1-1 encoding MR1-1-gCΔ. The MR1-1 sequence was inserted in-frame within gC replacing the a.a. residues 33 to 174 with MR1-1 to create the recombinant fusion protein MR1-1-gCΔ under the control of the gC promoter. This amplicon also bears the transgene cassette for GFP under the CMV promoter and the neo^R under the SV40 promoter. Abbreviations: SP, signal peptide; TM transmembrane domain; Circles, N-linked glycosylation site; a.a., position number. On the right panel, schematic representation of amplicon bearing MR1-1-gCΔ infecting glioma cells by specific binding between MR1-1 and EGFRvIII.

Fig. 2. Western blot of virions

293 cells were infected with hrR3 virus (lanes 1 and 2) or gC-minus virus (lanes 3 and 4), or were first transfected with pCONG-MR1-1 plasmid and then 24 hrs later infected with gC-minus virus (lanes 5 and 6) Virions were harvested and purified by density gradient centrifugation, and then virion proteins were resolved by SDS-PAGE and western blot analysis carried out with antibodies to gC (Panel A), gD (Panel B) or VP16 (Panel C) with stripping of signal between antibodies. In lanes 2, 4 and 6 virion proteins were treated with endo F prior to SDS-PAGE.

Fig. 3. Binding and infection efficiency of virions containing wild type and modified gC for U87 and U87ΔEGFR cells

Monolayers of U87 (solid) and U87ΔEGFR (open) glioma cells (1.5 × 10⁶ per well in 6-well plates) were incubated with different concentrations of virus stocks, including lane: 1) gCΔ2-3(gC-minus, lacZ+); 2) CONG-MR1-1 (gC modified to include MR1-1 in gCΔ2-3); 3) CONG-MRB in gCΔ2-3; 4) CONG-gC in gCΔ2-3 and hrR3 (gC+). Cells were infected with: 10³ tu/well corresponding to lacZ – in gCΔ2-3 or hrR3 (equivalent to M.O.I. = 0.015); in triplicate. Results represent the mean ± standard deviation of three independent experiments.

Fig. 4. Bioluminescence in tumors in vivo

Subcutaneous U87ΔEGFR tumors were injected with a combination of either MR1-gCΔ CONG + HRCFluc + gCΔ2-3 or gC CONG + HRCFluc + gCΔ2-3 vector stock and Luciferase activity was measured at 24 hrs, 48 hrs and 1 week after vector injection using bioluminescence imaging system. Left panel: The plotted values represent the average ratio between the photon counts per second measured that day and the photon counts measured on day 1 for each animal in tumor area ± S.D. Right panel: Representative pictures of bioluminescence emission from the U87ΔEGFR tumor of mice injected with amplicon vector carrying MR1-1-gCΔ (upper panel) or wild type gC (lower panel).